There are more than 40 million people infected by the HIV virus worldwide where 5 million new infections occurred during 2003. Worldwide, approximately 1 in every 100 adults aged 15 to 49 is infected with HIV. Although antiviral therapy can extend the life of individuals, the death toll continues to rise: 3 million people died from AIDS last year. Although antiviral nucleoside analog therapy successfully delays progression of HIV infection to AIDS, these drugs cause unwelcome side effects by inducing mitochondrial toxicity. Current antiviral nucleoside analog therapy against HIV results in compromised mitochondrial function due to selective inhibition of the mitochondrial DNA polymerase. As much as 20% of patients undergoing AZT treatment develop a mitochondrial dysfunctional disease known as red ragged fiber disease and D4T and ddC cause neuropathy in 15-20% of patients. The mode and effect of antiviral nucleotide analogs, by AZT, ddI, 3TC, D4T and others on the inhibition and fidelity of the mitochondrial DNA polymerase and mitochondrial DNA replication are poorly understood. What structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns is poorly understood. We previously evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase (pol gamma) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. Pol gamma has poor discrimination against many of the currently used NRTIs resulting in aborted DNA synthesis and subsequent depletion of mtDNA. Pol gamma readily incorporates ddCTP, ddITP and D4T-TP with an efficiency similar to the incorporation of normal nucleotides, whereas AZT-TP, CBV-TP, 3TC-TP and PMPApp act as moderate inhibitors to DNA synthesis. The structural elements responsible for this inhibition and the amino acids that interact with these analogs are unknown. Last year, we explored the role of three highly conserved amino acid residues in the active site of human pol gamma that modulate selection of nucleotide analogs as substrates for incorporation. Sequence alignments, crystal structures and mutagenesis studies of Family A DNA polymerases led us to change Tyr951 and Tyr955 in polymerase motif B to Phe and Ala, and Glu895 in polymerase motif A was changed to Ala. The mutant polymerases were tested for their ability to incorporate natural nucleotides and the five antiviral nucleoside analogs currently approved for antiviral therapy: AZT, ddC, D4T, 3TC and carbovir. To seek a three dimensional structural explanation for the unique selection for NRTIs by the human pol gamma a structural model of the human pol gamma was developed to ascertain the role of active site amino acids. One residue in particular, Y951 in motif B, is primarily responsible for the selection of dideoxynucleotides and D4T-TP. Our structural model of the human pol gamma should assist in rational design of antiviral nucleoside analogs with higher specificity for HIV-RT and minimal selection and incorporation into mitochondrial DNA.